In 1999 new cases of cancer of the prostate gland were expected to be diagnosed in 179,300 men in the U.S. and 37,000 American males were expected to die from this disease (Landis, S. H. et al. CA Cancer J. Clin. 49:8-31 (1999)). Prostate cancer is the most frequently diagnosed malignancy (other than that of the skin) in U.S. men and the second leading cause of cancer-related deaths (behind lung cancer) in that group.
Compositions useful in the treatment of prostatic cancer and related conditions are described in U.S. Pat. No. 5,948,750, issued Sep. 7, 1999 (corresponding to PCT Publ.No. WO 98/18493). Said compositions comprise chemical conjugates comprising known cytotoxic agents and oligopeptides having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen and that include a cyclic amino acid having a hydrophilic substituent. The oligopeptide moieties are selected from oligomers that are selectively recognized by free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity thereof.
Ideally, the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the intact oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly, or is restored completely, upon proteolytic cleavage of the attached oligopeptide at the cleavage site. Anthracycline antibiotics, in particular doxorubicin, are among the cytotoxic agents that were described in the published patent applications as preferably incorporated into such conjugates, which may be referred to as PSA conjugates. The PSA conjugates that incorporate doxorubicin that have been previously described incorporate the oligopeptide on the amine moiety of the sugar residue of doxorubicin. Those oligopeptides preferably incorporate a N-terminus protecting group to prevent or reduce proteolysis of the oligopeptide by non-PSA enzymes.
Among the preferred N-terminus protecting groups that are incorporated onto a PSA conjugate are the dicarboxylic acid alkanes, such as succinyl, glutaryl and the like. One of the preferred compounds described in U.S. Ser. No. 08/950,805, now U.S. Pat. No. 5,948,750 (PCT Publ.No. WO 98/18493) incorporating such an N-terminus protecting group is the compound of Formula 4: 
It is the object of this invention to provide a salt form of Compound 4 which is characterized by properties that offer advantages in the preparation, handling, storage and delivery of the compound to a patient in need of anti-cancer treatment.
It is the further the object of this invention to provide a stable lyophilized formulation of Compound 4 which is characterized by properties that offer advantages in the storage of the compound and delivery of the compound to a patient in need of anti-cancer treatment.
A sodium salt of a PSA conjugate compound having the formula 5 is disclosed: 
Such a salt is useful in the treatment of prostate cancer and benign prostatic hyperplasia (BPH).
Also described are formulations that comprise the salt of the invention and methods of preparing the salt.
The present invention is directed to the sodium salt of the formula 5: 
It has been surprisingly discovered that the sodium salt of a PSA conjugate compound, which is specifically described as the free acid in Example 4 of PCT Publ. No. WO 98/18493, is characterized by several advantageous physical properties when compared to the previously described free acid form (formula 4 hereinabove).
In particular, the sodium salt is crystalline and can be precipitated from an aqueous solution by the addition of a water miscible organic solvent. Such water miscible solvents include, but are not limited to tetrahydrofuran, methanol, ethanol, isopropanol and acetone. Preferably, acetone is utilized to precipitate the salt from solution. The crystalline nature of the sodium salt also allows for the purification of large quantities of the compound by recrystallization. Purification of the previously disclosed free acid requires the use of chromatographic techniques and freeze drying/lyophilization that are not amenable to large scale preparations which are often associated with commercial pharmaceutical agents.
It has also been surprisingly discovered that the sodium salt compound of the Formula 5 is more thermally stable than the corresponding free acid compound (Formula 4). An aqueous solution of the sodium salt of the instant invention has a pH of greater than 5.0. It has been discovered that the bond between the sugar moiety of doxorubicin and the tetracyclic doxorubicinone moiety is more readily cleaved at aqueous pH of less than 4.0. Therefore the sodium salt offers clear formulation advantages over the free acid, which has an unbuffered aqueous pH of less than 4.0.
It has further been discovered that the sodium salt of the Formula 5 offers advantages with respect to dissolution in water. The solubility of the sodium salt of the Formula 5 at room temperature is greater than 277 mg/mL of water. The solubility of the free acid (Compound 4) is 13.8 mg/mL of water at room temperature. It has also been surprisingly found that the sodium salt of the Formula 5 dissolves in water without forming aggregates, such as those that have been observed for the free acid. It has been found that the formation of aggregates hinders the filtration of an aqueous solution of the free acid compound through a 0.22xcexc filter, which is used to sterilize the aqueous solution prior to administration.
The instant invention is also directed to the process for the preparation of the salt of the Formula 5 which comprises the step of treating the acid of the Formula 4 with a base. The bases that may be used in the preparation of the salt of the Formula 5 include, but are not limited to: NaOH, Na2CO3, sodium acetate, sodium citrate (citric acid, trisodium salt) and the like. The preferred base is NaOH.
The instant invention is further directed to an alternative process for the preparation of the salt of the Formula 5 which comprises the step of treating the piperidine salt of the acid of the Formula 4 with a base. The bases that may be used in this preparation of the salt of the Formula 5 include, but are not limited to: NaOH, Na2CO3, sodium acetate, sodium citrate and the like. The preferred base is sodium acetate.
The instant invention is also directed to an second alternative process for the preparation of the salt of the Formula 5 which comprises the steps of a) treating the intermediate of the Formula 3 
with piperidine to provide a resulting mixture; b) treating the resulting mixture with an acid to provide a second resulting mixture; and c) treating the second resulting mixture with a base. The bases that may be used in this preparation of the salt of the Formula 5 include, but are not limited to: NaOH, Na2CO3, sodium acetate, sodium citrate and the like. The preferred base is sodium acetate. The acid that may be used in this preparation of the salt of the Formula 5 include, but are not limited to: hydrochloric acid and acetic acid. The preferred acid is acetic acid.
In an embodiment of the instant process, the process further comprises the step of precipitating the sodium salt of the Formula 5 from the resulting aqueous solution by adding a water miscible organic solvent to the solution. Such water miscible solvents include, but are not limited to, tetrahydrofuran, isopropanol and acetone. Preferably, acetone is utilized to precipitate the salt from solution.
In another embodiment of the instant process, the process further comprises the step of isolating the salt of the Formula 5 from the resulting aqueous solution by evaporating the solvent.
It has been surprisingly discovered that a lyophilized formulation of the PSA conjugate compound 4, which is specifically described in Example 4 of PCT Publ.No. WO 98/18493, is characterized by several advantageous physical properties when compared to the previously described free acid.
In particular, the lyophilized formulation is prepared by the addition of a salt of a carboxylic acid to an aqueous solution of the previously described free acid compound 4. The amount of carboxylate salt that is added to the prelyophilization solution of the compound of formula 4 is from about 3 molar equivalents of base of trisodium citrate per mole of compound 4 to about 9 molar equivalents of base per mole of compound 4. Preferably, the amount of salt that is added is about 5 molar equivalents of base per mole of compound 4. Lyophilization of this buffered solution provides a water-soluble solid that has improved storage stability.
Among the salts of a carboxylic acid that may be used to prepare the lyophilized formulation of the free acid compound 4 include but is not limited to: acetate, ascorbate, benzoate, citrate, formate, fumarate, lactate, maleate, malate, succinate tartarate-xcex1 and tartarate-m. Preferably the salt of carboxylic acid that is used is selected from citrate, succinate, tartrate-xcex1 and tartrate-m. The counterion of the salt may be sodium, potassium, lithium or calcium. Preferably, trisodium citrate is used to prepare the lyophilized formulation with the free acid compound 4. The amount of trisodium citrate that is added to the prelyophilization solution of the compound of formula 4 is from about 1 mole (3 molar equivalents of base) of trisodium citrate per mole of compound 4 to about 3 moles (9 molar equivalents) of trisodium citrate per mole of compound 4. Preferably, the amount of sodium citrate that is added is about 1.66 moles per mole of compound 4. Lyophilization of this buffered solution provides a water-soluble solid that has improved storage stability.
Alternatively, the lyophilized formulation is prepared by the addition of carboxylate/carboxylic acid buffer to an aqueous solution of the sodium salt compound 5, prior to lyophilization. Preferably, such a buffer comprises trisodium citrate/citric acid buffer, disodium succinate/succinic acid or sodium tartrate/tartaric acid. More preferably, the buffer comprises trisodium citrate/citric acid buffer. Preferably, the concentration of the sodium citrate/citric acid buffer is from about 10 mM to about 100 mM. More preferably, the concentration of the sodium citrate/citric acid buffer is from about 40 mM to about 60 mM. Preferably, the sodium citrate/citric acid buffer is from about pH 5.0 buffer to about pH 6.0 buffer.
It has been surprisingly discovered that the buffered formulation of the instant invention is more thermally stable than the lyophilized free acid compound of Formula 4, the crystalline salt compound 5 or an aqueous solution of either the free acid or salt compound. It has also been surprisingly discovered that the reconstituted solution of the lyophilized formulation of the instant invention is also more thermally stable than the aqueous solution of the free acid compound or the aqueous solution of the sodium salt compound 5.
It is preferred that the prelyophilization solution of either the PSA conjugate compound 4 or the salt compound 5 be at a pH that is from about 5.0 to about 6.0. More preferably, the pH of the prelyophilization solution is at a pH of from about 5.5 to about 6.0. Most preferably, the prelyophilization solution is at a pH of about 5.7. In order to maintain the prelyophilization solution at a preferred pH, an acid and/or a base is added to the buffered solution of compound 5 or compound 4. Preferably the acid that is added to the buffered solution is about 0.1 N HCl. Preferably, the base that is added to the buffered solution is about 0.1 N NaOH.
It has further been discovered that the lyophilized formulation offers advantages with respect to dissolution in water. The solubility of the free acid compound 4 at room temperature is directly dependent on the pH of the solution, from 3.98 mg/mL at pH 2.35 to greater than 156 mg/mL of water at pH 5.72.
It has also been surprisingly found that the instant lyophilized formulation dissolves in water without forming aggregates, such as those that have been observed for the free acid. It has been found that the formation of aggregates hinders the filtration of an aqueous solution of the free acid compound through a 0.22xcexc filter, which is used to sterilize the aqueous solution that is administered in the clinical setting.
It has also been discovered that addition of a sugar to the prelyophilization solution used to prepare the lyophilized formulation of the instant invention increases the stability of the lyophilized formulation and provides for a product having a longer pharmaceutical shelf-life. The added sugar may also act as a bulking agent and help reduce the hygroscopicity of the formulation. Thus, the instant formulation optionally further comprises a sugar, which is selected from glucose, mannitol, lactose, sucrose, fructose and the like. Preferably, the instant lyophilized formulation further comprises sucrose. Preferably the amount (in moles) of the sugar added to the prelyophilization solution used to prepare the lyophilized formulation is from about 2 times the amount (in moles) of compound 4 to about 20 times the amount (in moles) of compound 4 or compound 5. More preferably, the amount (in moles) of the sugar added to the prelyophilization solution used to prepare the lyophilized formulation is from about 5 times the amount (in moles) of compound 4 or compound 5 to about 15 times the amount (in moles) of compound 4 or compound 5.
The following abbreviations are utilized in the specification and tables to denote the indicated amino acids and moieties:
The PSA conjugate lyophilized formulation of the invention may additionally comprise pharmaceutically acceptable carrier, excipient or diluent. In this regard, see, e.g. Remington""s Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Company, edited by Osol et al. Such compositions may include proteins, such as serum proteins, for example, human serum albumin, and the like. Suitable diluents for reconstituting the lyophilized formulation prior to administration may include, for example, sterile water, isotonic saline, dilute aqueous dextrose, a polyhydric alcohol or mixtures of such alcohols, for example, glycerin, propylene glycol, polyethylene glycol and the like. As used, xe2x80x9cpharmaceutically acceptablexe2x80x9d refers to those agents which are useful in the treatment or diagnosis of a warm-blooded animal including, for example, a human, equine, porcine, bovine, murine, canine, feline, or other mammal, as well as an avian or other warm-blooded animal. The preferred mode of administration of the reconstituted formulation is parenterally, particularly by the intravenous, intramuscular, subcutaneous, intraperitoneal, or intralymphatic route.
As used herein, the terms xe2x80x9ccompositionxe2x80x9d and xe2x80x9cformulationxe2x80x9d are intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specific ingredients.
The formulation of the instant invention may also be administered in combination with an inhibitor of prenyl-protein transferase, in particular farnesyl-protein transferase.
For intravenous administration, the composition preferably will be prepared so that the amount administered to the patient will be from about 0.01 to about 1 g of the conjugate. Preferably, the amount administered will be in the range of about 0.2 g to about 1 g of the conjugate. The salt of the invention is effective over a wide dosage range depending on factors such as the disease state to be treated or the biological effect to be modified, the manner in which the conjugate salt is administered, the age, weight and condition of the patient as well as other factors to be determined by the treating physician. Thus, the amount administered to any given patient must be determined on an individual basis.